Q Beta (Q.beta.) replicase is a template-specific RNA-directed RNA polymerase derived from the bacteriophage Q.beta.. In vivo, the normal function of Q.beta. replicase is to replicate the RNA genome of the Q.beta. bacteriophage to produce progeny phage genomes. Each infectious Q.beta. virion contains one molecule of single stranded RNA of molecular weight 1.5.times.10.sup.6, which is termed the viral plus (+) strand. This is the strand utilized as mRNA to direct viral protein synthesis. Using the (+) strand as a template, Q.beta. replicase produces an RNA copy of the template which is complementary to the original template. These RNA molecules are termed minus (-) strands. Importantly, both the (+) and (-) strands are templates for the enzyme. Therefore, replication of the RNA template proceeds in a geometric fashion (i.e., 1, 2, 4, 8, 16, 32, etc.).
In vitro, the enzyme can utilize a limited number of other RNA molecules besides the Q.beta. genome as templates. One such RNA template which has been relatively well studied is termed midivariant (MDV). MDV is significantly smaller than the Q.beta. RNA genome and was discovered as a naturally occurring product in Q.beta. replicase reactions. Significant effort has been invested in designing variants of MDV RNA which can serve as amplifiable reporter probes in nucleic acid hybridization assays.
In general terms, a reporter probe will serve two functions. It will contain a nucleotide sequence which permits it to hybridize specifically with a predetermined target nucleic acid, and it will contain a ligand of some sort which permits its detection in an assay. Common detection ligands are radioactive P-32 or I-125, fluorescein, or biotin which can be coupled to probe sequences in a variety of ways.
In the case of MDV reporter probes, a probe sequence is built into the MDV molecule in such a way that it: 1) permits the MDV probe to specifically hybridize to its intended target nucleic acid, and 2) remains replicatable by Q.beta. replicase in spite of the additional probe sequence. Thus, the MDV serves as an amplifiable detection ligand. One billion or more progeny molecules can be produced from a single starting template MDV molecule in approximately 30 minutes. Thus, a very large number of detection ligands (MDV RNA molecules) can be produced from very few hybridized reporter probes. This permits the development of extremely sensitive nucleic acid hybridization assays; that is, assays which are capable of detecting the presence of very few target molecules (or organisms) in a test sample.
A variety of engineered MDV probe molecules are described in U.S. Pat. No. 4,786,600, and patent applications Ser. Nos. 252,243 and 370,218, the teachings of which are incorporated herein by reference.
Assay sensitivity is a function not only of the amount of signal that can be generated for a given amount of target nucleic acid, but also of the amount of "background" signal which is generated even in the absence of target nucleic acid. One significant source of background signal in nucleic acid probe systems using Q.beta. replicase is the presence in some Q.beta. replicase preparations of contaminating RNA termed "wild-type" or "endogenous" variant RNA. Q.beta. replicase prepared by the procedure of Eoyang and August typically contains between 100 and 10,000 molecules of wild type variant RNA per microgram of protein. Eoyang and August, Prog. Nucl. Acids Res., 2:829-839 (1972). This wild type variant RNA replicates and generates a signal even when exogenous (i.e., probe) template RNA is omitted from the reaction. This wild type variant RNA competes with the probe RNA for Q.beta. replicase, severely limiting the enzyme's ability to replicate low numbers of probe RNAs to detectable levels. This, in turn, limits the achievable sensitivity of the assay to some value above the background level of wild type variant RNA.
One means of limiting this background signal would be to improve the purification of the Q.beta. replicase enzyme so that it contains less (or no) contaminating wild type variant RNA. DiFrancesco, in co-pending patent application U.S. Ser. No. 07/364,306, filed Jun. 9, 1989 which is incorporated herein by reference, discloses an improved Q.beta. replicase purification protocol which yields highly purified enzyme that appears to be free of wild type MDV RNA.
Another means of limiting assay background due to wild type MDV would be to devise recombinant MDV probe molecules and assay conditions that enhance the replication of probe molecules relative to that of wild type MDV molecules. Kramer et al., J. Mol. Biol. 89:719-736 (1974) describe a mutant MDV RNA whose replication is less sensitive than "wild type" (endogenous) MDV to the dye ethidium bromide. However, Kramer et al. do not discuss the use of such mutant MDV molecules as probes,
A major consideration in the convenience and precision of assays employing Q.beta. replicase is detection of the amplified products. In vitro, Q.beta. replicase is capable of replicating one MDV molecule. However, it also is a property of Q.beta. replicase that the time course of the appearance of replication products reflects the amount of MDV template (or probe) present at the beginning of the reaction. That is, given a particular set of reaction conditions, a larger amount of input MDV will yield observable levels of product MDV sooner than lower input levels. In fact, there is a predictable, mathematical relationship between the input MDV and the time to observable product. Thus, there is significant value in monitoring the kinetics of the Q.beta. replicase reaction in that information about the level of target nucleic acid in the experimental sample can be inferred.
By careful selection of the inhibitory chemical agent, MDV probes can be designed which permit a convenient, quantitative, kinetic analysis of MDV production during the replication (amplification) reaction.